Panel A shows sets of microscopy images depicting embryos stained with anti-HA and anti-Vasa to confirm expression of dp110 constructs and stain PGCs. Each set includes three images: one for dp110 (αHA), one for Vasa, and a merged image with DAPI. Panel B contains a scatter plot showing the number of PGCs per embryo for different genotypes, with axes labeled as PGCs/embryo and genotype. The plot includes statistical annotations indicating significance levels. Below the scatter plot, two microscopy images depict embryos from gcl knockout mothers expressing RNAi against dp110, stained with Vasa and DAPI. Panel C features another scatter plot showing the number of PGCs per embryo for gcl homozygous mutant mothers expressing RNAi against dp110, with axes labeled similarly to Panel B. Panel D presents a schematic diagram of dp110-WT and variant constructs, highlighting different domains and mutations.
PI3K strongly antagonizes PGC formation through its Ras-binding and catalytic activity. (A) Embryos from mothers overexpressing germplasm-targeted, HA-tagged dp110-WT, dp110-CAAX (constitutively active), dp110-D954A (catalytically inactive), and dp110-ΔRBD (no Ras binding) were immunostained with anti-HA and anti-Vasa to confirm expression of the construct and stain PGCs for counting. Maternal expression of the UASp constructs was driven by two copies of maternal-tubulin-GAL4-VP16 for optimal expression during late oogenesis. Images depict maximum intensity projections spanning the area of PGC formation. Scale bar = 50 µm. (B) PGCs in nuclear cycle 13–14 embryos from mothers of the indicated genotype were counted and plotted from A. The genotype of control mothers is maternal-tubulin-GAL4-VP16/+; maternal-tubulin-GAL4-VP16/+. Embryos from control, dp110-D954A, and dp110-ΔRBD mothers show no significant difference in PGC amounts. Bars represent the mean ± standard deviation. (n > 10, Mann–Whitney test). (C) Embryos from gcl−/− mothers expressing RNAi against dp110 were immunostained with anti-Vasa to count PGCs. Images depict maximum intensity projections spanning the area of PGC formation. Scale bar = 50 µm. (D) PGCs in nuclear cycle 13–14 embryos from mothers of the indicated genotype were counted and plotted from C. RNAi against mCherry was used as a control. Bars represent the mean ± standard deviation. (n > 10, Mann–Whitney test). (E) Schematic depicting dp110-WT and variant constructs used. All constructs contain the germplasm-targeting 3′UTR (TCEp3).